Nicking and extension amplification reaction (near) of streptococcus species

ABSTRACT

This invention relates to compositions, methods and kits for detecting the presence or absence of a bacterial species in a biological sample using isothermal nucleic acid amplification.

CLAIM OF PRIORITY

This application is a divisional of U.S. application Ser. No.16/417,071, filed May 20, 2019, which is a continuation of U.S.Application Ser. No. 15/391,002, filed Dec. 27, 2016, now U.S. Pat. No.10,329,601, issued Jun. 25, 2019, which claims the benefit of U.S.Provisional Patent Application Ser. No. 62/271,400, filed on Dec. 28,2015; the entire contents of each of which is hereby incorporated byreference.

TECHNICAL FIELD

This invention relates to methods and compositions for detecting thepresence or absence of a bacterial species in a biological sample usingisothermal nucleic acid amplification. More specifically, the presentinvention relates to using Nicking and Extension Amplification Reaction(NEAR) to detect Streptococcus pyogenes (GAS or Strep A) in a biologicalsample.

BACKGROUND

Certain isothermal amplification methods are able to amplify a targetnucleic acid from trace levels to very high and detectable levels withina matter of minutes. Such isothermal methods, e.g., Nicking andExtension Amplification Reaction (NEAR), allow users to detect aparticular nucleotide sequence in trace amounts, facilitatingpoint-of-care testing and increasing the accessibility and speed ofdiagnostics.

Streptococcus pyogenes is the causative agent of group A streptococcal(GAS) infections such as pharyngitis, impetigo, and life-threateningnecrotizing fasciitis and sepsis. The most common GAS infection,pharyngitis, can be diagnosed by collecting a throat swab sample from apatient and culturing the sample under conditions that would enablebacterial, specifically S. pyogenes, growth, which takes 2-3 days.Culturing S. pyogenes is an accurate and reliable method of diagnosingGAS, but it is slow. A 2-3 day delay in prescribing appropriateantibiotic treatment can result in unnecessary patient suffering andpotentially the onset of life threatening conditions such as rheumaticfever. In the recent past, biochemical methods have been developed todetect S. pyogenes, but these methods do not provide the necessarycharacteristics to be deployed in the point-of-care setting, either dueto a lack of sensitivity or time to result (speed).

Accordingly, a highly sensitive and rapid qualitative assay for thedetection and diagnosis of a S. pyogenes infection is desired.

SUMMARY

This disclosure is based, at least in part, on the discovery that thepresence or absence of S. pyogenes (GAS or Strep A) in a biologicalsample can be accurately and efficiently detected in using the Nickingand Extension Amplification Reaction (NEAR). In view of this discovery,provided herein are NEAR compositions and methods for detecting thepresence or absence of a S. pyogenes in a biological sample. Thecompositions provided herein are useful for the detection of S. pyogenesnucleic acid in a biological sample, and comprise at least one pair oftemplates (i.e., a forward and reverse template pair), and optionally aprobe, specific for S. pyogenes.

In one aspect, this disclosure features compositions that include aforward template comprising a nucleic acid sequence comprising arecognition region at the 3′ end that is complementary to the 3′ end ofthe S. pyogenes target nucleic acid sequence, a nicking enzyme bindingsite and a nicking site upstream of said recognition region, and astabilizing region upstream of said nicking site; and a reverse templatecomprising a nucleotide sequence comprising a recognition region at the3′ end that is complementary to the 3′ end of the S. pyogenes targetnucleic acid sequence, a nicking enzyme binding site and a nicking siteupstream of said recognition region, and, a stabilizing region upstreamof said nicking site. The portion of the template that is complementaryto the target nucleotide sequence can be 8-30 nucleotides in length,8-25 nucleotides in length, 8-20 nucleotides in length, or 8-15nucleotides in length.

In some embodiments, S. pyogenes target nucleic acid sequence is foundwithin the S. pyogenes cell envelope proteinase A (cepA) gene sequence.Accordingly, the forward template comprises a recognition region at the3′ end that is complementary to the 3′ end of the S. pyogenes cepA geneantisense strand, and the reverse template comprises a recognitionregion at the 3′ end that is complementary to the 3′ end of the S.pyogenes cepA gene sense strand. Accordingly, the portion of therecognition region that is complementary to the 3′ end of the targetantisense strand is 8-30 nucleotides in length and the recognitionregion that is complementary to the 3′ end of the target sense strand is8-30 nucleotides in length.

In some embodiments, the compositions disclosed herein further comprisean oligonucleotide probe comprising a sequence complementary to the S.pyogenes target nucleotide sequence (e.g., the S. pyogenes cepAnucleotide sequence). In some embodiments of all aspects, thecomposition includes a probe labeled with a detectable label. In someembodiments, the detectable label is a fluorophore, an enzyme, aquencher, an enzyme inhibitor, a radioactive label, an electrochemicallabel, a chemiluminescent label, a metal particle, a latex particle, onemember of a binding pair or any combination thereof.

In some embodiments of any of the aspects described here, the targetnucleic acid sequence is also combined with a probe labeled with adetectable label. In some embodiments of all aspects, the probecomprises an oligonucleotide complimentary to a portion of the targetnucleic acid sequence at a position that is in between the portions ofthe target nucleic acid sequence that are complementary to the first andthe second templates.

In some embodiments, the portion of the nucleic acid sequence that iscomplementary to the 3′ end of the target antisense strand is 8-30nucleotides in length.

This disclosure also provides methods for detecting the presence orabsence of S. pyogenes in a biological sample by identifying S. pyogenesnucleic acid in the sample. In some aspects, this disclosure featuresmethods for the detection of the presence or absence of S. pyogenes in abiological sample, the methods comprising contacting the biologicalsample with components of a nucleic amplification reaction for a S.pyogenes target nucleic acid sequence comprising i) a forward templatecomprising a nucleic acid sequence comprising a recognition region atthe 3′ end that is complementary to the 3′ end of the S. pyogenes targetnucleic acid sequence; a nicking enzyme binding site and a nicking siteupstream of said recognition region, and a stabilizing region upstreamof said nicking site, ii) a reverse template comprising a nucleotidesequence comprising a recognition region at the 3′ end that iscomplementary to the 3′ end of the S. pyogenes target nucleic acidsequence; a nicking enzyme binding site and a nicking site upstream ofsaid recognition region, and, a stabilizing region upstream of saidnicking site, (iii) a first nicking enzyme that is capable of nicking atthe nicking site of said forward template, and does not nick within saidtarget sequence, (iv) a second nicking enzyme that is capable of nickingat the nicking site of said reverse template and does not nick withinsaid target sequence, and (v) a DNA polymerase; amplifying the targetnucleic acid sequence in a nucleic amplification reaction to provide anamplification product; and determining (e.g., detecting) the presence orabsence of the amplification product.

According to any embodiment of the methods provided herein,amplification is performed by multiple cycles of said polymeraseextending said forward and reverse templates along said target sequenceproducing a double-stranded nicking site, said nicking enzymes nickingat said nicking sites, and, following a first round of amplification, atamplified copies of said sites, producing an amplification product,thereby providing a product. In some embodiments, amplifying the targetnucleic acid sequence comprises performing an isothermal nucleicamplification reaction. Such isothermal methods, include NEAR andRecombinase Polymerase Amplification (RPA) methods.

In some embodiments, the present amplification methods do not requirethe use of temperature cycling (i.e., the present amplification methodsare performed isothermally), as often is required in methods ofamplification to dissociate the target sequence from the amplifiednucleic acid. The temperature of the reaction may vary based on thelength of the sequence, and the GC concentration, but, as understood bythose of ordinary skill in the art, the temperature should be highenough to minimize non-specific binding. The temperature should also besuitable for the enzymes of the reaction, the nicking enzyme and thepolymerase. For example, the reaction may be run at about 52° C., 53°C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., or 60° C. In someembodiments, the reaction is run at about 37° C.-85° C., 37° C.-60° C.,54° C.-60° C., 55° C.-60° C., 58° C.-60° C. and, in exemplaryembodiments, from 56° C.-58° C. In certain embodiments, there is nodenaturation step in the process. The entire amplification process,including interacting templates with target nucleic acid, is conductedwithin substantially isothermal conditions, and without a denaturingstep (e.g., no significant temperature increase (e.g., no increase intemperature to 90-110° C.)), in some embodiments of the present methods.

In some embodiments of all aspects, the nucleic acid amplificationreaction is NEAR. In some embodiments of all aspects, the monitoring ofthe rate of increase of nucleic acid amplification products in themixture is performed in real-time.

According to any embodiment of the methods provided herein, determining(e.g. detecting) is performed using real-time fluorescence detection.

In some embodiments of any of the aspects described here, the targetnucleic acid is genomic DNA. In some embodiments of all aspects, thetarget nucleic acid is double-stranded or single-stranded nucleic acidmolecules, such as DNA (e.g., cDNA, gDNA, mtDNA, etc.) or RNA (e.g.,vRNA, mRNA, snRNA, rRNA, tRNA, etc.).

In some embodiments, the S. pyogenes target nucleic acid sequence isfound within the S. pyogenes cell envelope proteinase A (cepA) genesequence. Thus, in some aspects, this disclosure features methods forthe detection of the presence or absence of S. pyogenes in a biologicalsample, the methods comprising contacting a biological sample withcomponents of a nucleic amplification reaction for a S. pyogenes targetnucleic acid sequence comprising i) a forward template comprising anucleic acid sequence comprising a recognition region at the 3′ end thatis complementary to the 3′ end of the S. pyogenes cepA gene antisensestrand; a nicking enzyme binding site and a nicking site upstream ofsaid recognition region; and a stabilizing region upstream of saidnicking site; ii) a reverse template comprising a nucleotide sequencecomprising a recognition region at the 3′ end that is complementary tothe 3′ end of the S. pyogenes cepA gene sense strand; a nicking enzymebinding site and a nicking site upstream of said recognition region; anda stabilizing region upstream of said nicking site; (iii) a firstnicking enzyme that is capable of nicking at the nicking site of saidforward template, and does not nick within said target sequence; (iv) asecond nicking enzyme that is capable of nicking at the nicking site ofsaid reverse template and does not nick within said target sequence; and(v) a DNA polymerase; amplifying the target nucleic acid sequence in anucleic amplification reaction to provide an amplification product; anddetermining whether an indicator of the target nucleic acid species ispresent in the amplification product.

In some embodiments of all aspects, the components of a nucleicamplification reaction further comprise a probe oligonucleotidecomprising a sequence complementary to the S. pyogenes cell envelopeproteinase A (cepA) gene nucleotide sequence.

In some embodiment of all aspects, the nucleic amplification reaction isperformed under essentially isothermal conditions.

The amplification may be, for example, conducted at a constanttemperature. This temperature may be, for example, between 54° C. and60° C. As to the length of time for the reaction to take place, incertain examples, the amplification reaction is held at constanttemperature for 1 to 10 minutes.

In some embodiments of all aspects, the compositions disclosed hereincomprise reagents suitable for NEAR amplification of a target sequence.In some embodiments of all aspects, the methods disclosed hereincomprise combining a target nucleic acid with reagents suitable for NEARamplification and performing NEAR amplification.

In some embodiments of the compositions and methods disclosed herein,specific probes and templates for detecting S. pyogenes are provided.Representative template sequences include a forward template comprisinga nucleotide sequence having at least 80, 85 or 95% identity to SEQ IDNO: 1 (AGACTCCATATGGAGTCTAGCCAAACAGGAACA), and a reverse templatecomprising a nucleotide sequence having at least 80, 85 or 95% identityto SEQ ID NO 2: (CGACTCCATATGGAGTCGAAAGCAATCTGAGGA); or a forwardtemplate comprising a nucleotide sequence having at least 80, 85, or 95%identity to SEQ ID NO: 8: (AGACTCCACACGGAGTCTAGCCAAACAGGAACA), and areverse template comprising a nucleotide sequence having at least 80,85, or 95% identity to SEQ ID NO: 9:(GGACTCCACACGGAGTCCGCCAGCAATCUGAGG). A representative probeoligonucleotide includes a nucleotide sequence having at least 80, 85 or95% identity to SEQ ID NO: 3 (ACAAGTATGTGAGGAGAGGCCATACTTGT). In someembodiments of the compositions and methods described herein, thetemplates include a 3′ or 5′ modification.

In some embodiments of any of the aspects described here, thecompositions disclosed herein further comprise one or more of a DNApolymerase, one or more nicking enzymes, dNTPs or a mixture of dNTPs andddNTPs. The DNA polymerase can be selected from the group consisting ofGeobacillus bogazici DNA polymerase, Bst (large fragment), exo-DNAPolymerase, Manta 1.0 DNA Polymerase (Enzymatics ®).

The nicking enzyme may, for example, nick upstream of the nicking enzymebinding site, or, in exemplary embodiments, the nicking enzyme may nickdownstream of the nicking enzyme binding site. In certain embodiments,the forward and reverse templates comprise nicking sites recognized bythe same nicking enzyme and said first and said second nicking enzymeare the same. The nicking enzyme (“NE”) may, for example, be selectedfrom the group consisting of Nt.BspQI, Nb.BbvCi, Nb.BsmI, Nb.BsrDI,Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Bpu10I, Nt.Bpu10Iand N.BspD61.

In some embodiments of any of the aspects described here, thecomposition further comprises a lytic agent, such as a bacteriophagelysin. In some embodiments the lytic agent is a bacteriophage lysincomprising streptococcal C1 bacteriophase lysin (PlyC).

In some embodiments of any of the aspects described here, thecomposition is a lyophilized composition.

In some embodiments of all aspects, the templates and/or probes can belabeled with (e.g., conjugated to) a detectable label. Representativedetectable labels include a fluorophore, an enzyme, a quencher, anenzyme inhibitor, a radioactive label, a member of a binding pair, andcombinations thereof. In some embodiments, the detectable label is afluorescent moiety suitable for use in real-time nucleic amplificationreactions. Such fluorescent moieties are known to persons skilled in theart and are available from various commercial sources.

In some embodiments of any of the aspects described here, the templatesand/or probes can comprise comprises one or more modified nucleotides,spacers, or blocking groups. Representative modified nucleotides,spacers, or blocking groups include a 2′modification, a 2′-O-methyl, orat least one phosphorothioate. In some embodiments of any of the aspectsdescribed herein, the templates and/or probes can comprise a 2-Ome(2′-O-methyl) modification one or more (e.g., 1, 2, 3, 4, 5 or more)bases of the template and/or probe. For example, SEQ ID NO: 8 caninclude a 2′-O-methyl modification on five bases at the 3′ end(AGACTCCACACGGAGTCTAGCCAAACAGmGmAmAmCmA) or SEQ ID NO:9 can include a2′-O-methyl modification on five bases at the 3′ end(GGACTCCACACGGAGTCCGCCAGCAATCmUmGmAmGmG).

In some aspects, the disclosure provides kits containing templates andprobes for the detection of S. pyogenes in a biological sample andcomponents of a nucleic acid amplification reaction, such as apolymerase. Representative template sequences include a forward templatecomprising a nucleotide sequence having at least 80, 85 or 95% identityto SEQ ID NO: 1 (AGACTCCATATGGAGTCTAGCCAAACAGGAACA); a reverse templatecomprising a nucleotide sequence having at least 80, 85 or 95% identityto SEQ ID NO 2: (CGACTCCATATGGAGTCGAAAGCAATCTGAGGA); and a probeoligonucleotide comprising a nucleotide sequence at least 80, 85 or 95%identity to SEQ ID NO: 3 (ACAAGTATGTGAGGAGAGGCCATACTTGT). Representativetemplate sequences also include a forward template comprising anucleotide sequence having at least 80, 85 or 95% identity to SEQ ID NO:8 (AGACTCCACACGGAGTCTAGCCAAACAGGAACA); a reverse template comprising anucleotide sequence having at least 80, 85 or 95% identity to SEQ ID NO:9 (GGACTCCACACGGAGTCCGCCAGCAATCUGAGG); and a probe oligonucleotidecomprising a nucleotide sequence at least 80, 85 or 95% identity to SEQID NO: 3 (ACAAGTATGTGAGGAGAGGCCATACTTGT).

In some embodiments, the kit contains instructions to use the kit. Insome embodiments, the kits contain a swab for obtaining a biologicsample, dNTPs or a mixture of dNTPs and ddNTPs, and a lytic agent. Insome embodiments, the kit comprises reagents for gaining access toand/or extracting/isolating nucleic acid from a biological sample.

The kit may, for example, provide said polymerase, nicking enzymes, andtemplates in a container. The kit may provide, for example, saidpolymerase, nicking enzymes, and templates in two containers. In certainexamples, the polymerase and nicking enzymes are in a first container,and said templates are in a second container. In certain examples, thepolymerase and nicking enzymes are lyophilized. The kit may, forexample, further comprise instructions for following the amplificationmethods of the present invention. The kit may, for example, furthercomprise a lateral flow device or dipstick. The lateral flow device ordipstick may, for example, further comprise a capture probe, whereinsaid capture probe binds to the amplified product. The kit may, forexample, further comprise a detector component, for example, oneselected from the group consisting of a fluorescent dye, colloidal goldparticles, latex particles, a molecular beacon, polystyrene beads, andthe like. In other examples, at least one of the templates of the kitmay comprise a spacer, blocking group, or modified nucleotides.

The term “template” or “primer” refers to an oligonucleotide that iscapable of acting as a point of initiation for the 5′ to 3′ synthesis ofa primer extension product that is complementary to a nucleic acidstrand. The primer extension product is synthesized in the presence ofappropriate nucleotides and an agent for polymerization, such as a DNApolymerase, in an appropriate buffer and at a suitable temperature.

As used herein, the term “probe” refers to an oligonucleotide that formsa hybrid structure with the product generated by amplification of thetarget nucleic acid sequence in a sample undergoing analysis, due tocomplementarity of at least one sequence in the probe with the targetsequence. The nucleotides of any particular probe may bedeoxyribonucleotides, ribonucleotides, and/or synthetic nucleotideanalogs.

Within the context of the present invention, the target nucleic acidsequence is a nucleic acid sequence of S. pyogenes, i.e., GAS or StrepA.

The term “one or more” or “at least one” as used in the presentinvention stands for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 compound(s) or evenmore.

The terms “first” and “second” are used in this disclosure in theirrelative sense only. It will be understood that, unless otherwise noted,those terms are used merely as a matter of convenience in thedescription of one or more of the embodiments. The terms “first” and“second” are only used to distinguish one element from another element,and the scope of the rights of the disclosed technology should not belimited by these terms. For example, a first element may be designatedas a second element, and similarly the second element may be designatedas the first element.

The terms “increased”, “increase” or “up-regulated” are all used hereinto generally mean an increase by a statistically significant amount; forthe avoidance of any doubt, the terms “increased” or “increase” means anincrease of at least 10% as compared to a reference level, for examplean increase of at least about 20%, or at least about 30%, or at leastabout 40%, or at least about 50%, or at least about 60%, or at leastabout 70%, or at least about 80%, or at least about 90% or up to andincluding a 100% increase or any increase between 10-100% as compared toa reference level, or at least about a 0.5-fold, or at least about a1.0-fold, or at least about a 1.2-fold, or at least about a 1.5-fold, orat least about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 1.0-fold and 10-fold or greater as compared to areference level.

The terms “decrease”, “decreased”, “reduced”, “reduction” or“down-regulated” are all used herein generally to mean a decrease by astatistically significant amount. However, for avoidance of doubt,“reduced”, “reduction”, “decreased” or “decrease” means a decrease by atleast 10% as compared to a reference level, for example a decrease by atleast about 20%, or at least about 30%, or at least about 40%, or atleast about 50%, or at least about 60%, or at least about 70%, or atleast about 80%, or at least about 90% or up to and including a 100%decrease (i.e. absent level as compared to a reference sample), or anydecrease between 10-100% as compared to a reference level, or at leastabout a 0.5-fold, or at least about a 1.0-fold, or at least about a1.2-fold, or at least about a 1.5-fold, or at least about a 2-fold, orat least about a 3-fold, or at least about a 4-fold, or at least about a5-fold or at least about a 10-fold decrease, or any decrease between1.0-fold and 10-fold or greater as compared to a reference level.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the described subject matter inany way. When definitions of terms in incorporated references appear todiffer from the definitions provided in the present teachings, thedefinition provided in the present teachings shall control. It will beappreciated that there is an implied “about” prior to metrics such astemperatures, concentrations, and times discussed in the presentteachings, such that slight and insubstantial deviations are within thescope of the present teachings herein. In this application, the use ofthe singular includes the plural unless specifically stated otherwise.Also, the use of “comprise,” “comprises,” “comprising,” “contain,”“contains,” “containing,” “include,” “includes,” and “including” are notintended to be limiting. It is to be understood that both the foregoinggeneral description and the following detailed description are exemplaryand explanatory only and are not restrictive of the invention. Thearticles “a” and “an” are used herein to refer to one or to more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one element or more than one element.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and are not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the results of an exemplary limit of detection (LOD) studyperformed comparing four mixes containing the target assay (25C—completeassay, 4× 25 μl lyo pellet; 25T—target only assay, 4× 25 μl lyo pellet;50C—complete assay, 2× 50 μl lyo pellet; 50T—target only assay, 2× 50 μllyo pellet). Reactions were performed on the Stratagene Mx3005P thermalcycler using a standard ‘hot start’ approach.

FIG. 2 shows the results of an exemplary LOD study performed comparingfour mixes (25C—complete assay, 4× 25 μl lyo pellet; 25T—target onlyassay, 4× 25 μl lyo pellet; 50C—complete assay, 2× 50 μl lyo pellet;50T—target only assay, 2× 50 μl lyo pellet). All reactions wereperformed on the Stratagene Mx3005P thermal cycler using a ‘hot start’approach.

FIG. 3 shows the results of an exemplary GAS IC assay (25I & 50I)performed in the presence of increasing amounts of GAS genomic DNA toassess the impact of this inhibitor on target assay performance.

FIG. 4 depicts a typical wet GAS NEAR assay performed in the presence of100 copies of inactivated GAS bacteria with or without 1 μg of plyC.Legend: pur. C-His plyC—C-terminus His-tagged plyC; controlplyC—non-tagged plyC.

FIG. 5 shows the results of a GAS NEAR assay performed to determine thelowest copy number at which the assay could reproducibly detect targetnucleic acid (requirement of detecting 95% of replicates). Shown are theresults from the 50T mix when tested at 25° C. without a pre-heatingstep.

FIG. 6 shows the impact of human gDNA on the GAS assay. Lyophilized GASNEAR assays were performed with 0, 25, 250 or 1,000 copies of target GASgDNA in the presence of increasing amounts of background human gDNA (0to 1,000 ng). Mixes 50I (0 copies of target; lower right panel) and 50T(25 copies (upper left panel), 250 copies (upper right panel) or 1,000copies (lower left panel) of target gDNA) were screened on theStratagene Mx3005P instrument using a typical hot start approach.

DETAILED DESCRIPTION

This disclosure is based in part on the discovery that it is possible todetect the presence or absence of S. pyogenes in a biological sampleusing isothermal amplification. To that end, the present applicationdiscloses a composition for detecting the presence or absence of S.pyogenes in a biological sample by amplifying and detecting a targetnucleotide sequence using NEAR coupled with a real-time fluorescence(e.g., real-time PCR). In some embodiments, the target nucleotidesequence can be detected using real-time fluorescence approaches.

The use of the term “target sequence” may refer to either the sense orantisense strand of the sequence, and also refers to the sequences asthey exist on target nucleic acids, amplified copies, or amplificationproducts, of the original target sequence. The amplification product maybe a larger molecule that comprises the target sequence, as well as atleast one other sequence, or other nucleotides.

Methods of this invention can be used to identify nucleic acid fromspecimens for diagnosis of S. pyogenes infection. The specific primersand probes of the invention that are used in these methods allow for theamplification of and monitoring the development of specificamplification products. The increased sensitivity of NEAR for detectionof S. pyogenes as well as the improved features of real-timefluorescence including sample containment and real-time detection of theamplified product, make feasible the implementation of this technologyfor routine diagnosis of S. pyogenes infections in the clinicallaboratory and at point-of-care.

Certain isothermal amplification methods are able to amplify targetnucleic acid from trace levels to very high and detectable levels withina matter of minutes. Such isothermal methods, e.g., NEAR and RPA canallow users to detect a particular sequence in trace amounts,facilitating point-of-care testing and increasing the accessibility andspeed of diagnostics.

The time that the amplification reaction is run may vary from, forexample, within about 1 minute, or within about 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 minutes. Longer reactiontimes may produce acceptable results where speed is not an issue. Insome embodiments, the reaction is between 1-20 minutes, 1-15 minutes or1-10, 1-8, 1-5, 1-2.5, 2.5-5, 2.5-8, 2.5-10, or 2.5-20 minutes incertain embodiments. The amplification processes described herein areefficient, and in some embodiments, there is about 1×10⁶-fold or moreamplification, about 1×10⁷-fold or more amplification, about 1×10⁸-foldor more amplification, about 1×10⁹-fold or more amplification, or about1×10¹⁰-fold or more amplification in the time frame of the reaction, forexample, in 5, 10 or twelve minutes. The reaction is highly sensitive,and is able to detect, for example, as low as about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 copies, or more, in asample, as many as 200, 500, 1,000, 5,000, or 10,000, or more copies ina sample, or, for example, may detect a target that is present at aconcentration of, for example, about 3.32E-13 micromolar to about3.32E-8 micromolar, about 1.66E-12 micromolar to about 3.32E-8micromolar, about 3.32E-13 micromolar to about 3.32E-7 micromolar, orabout 3.32E 13 micromolar to about 3.32E-6 micromolar.

NEAR methods are disclosed, e.g., in U.S. 2009/0081670 and U.S.2009/0017453 each of which are incorporated herein by reference.

The isothermal nature of NEAR, coupled with the production of shortproducts and geometric amplification of either DNA or RNA targets,provides an ultra-rapid amplification method. As disclosed herein,coupling of this approach to a real-time fluorescence detection approachprovides exquisite sensitivity and specificity. Further, NEAR isdistinguished from most molecular tests as there is no need for alengthy or cumbersome sample preparation/nucleic acid purification.These key attributes make NEAR an ideal technology for point-of-care(POC) integration, where a rapid and reliable result is essential. Basedon these principles, the present disclosure provides a NEAR assay todetect the presence of the bacterium Streptococcus pyogenes (GAS orStrepA) when present in a biological sample, e.g., a clinical throatswab sample.

Nucleic acids (e.g., polynucleotides) suitable for amplification inconnection with the present methods include double-stranded andsingle-stranded nucleic acid molecules, such as DNA and RNA molecules.The polynucleotides may be of genomic, chromosomal, plasmid,mitochondrial, cellular, and viral nucleic acid origin. For doublestranded polynucleotides, the amplification may be of either one or bothstrands.

Another such isothermal amplification method suitable for the methodsdisclose herein is Recombinase Polymerase Amplification (RPA). RPA canallow users to detect a particular sequence in trace amounts,facilitating point-of-care testing and increasing the accessibility andspeed of diagnostics. As described here, RPA employs enzymes, known asrecombinases, that are capable of pairing oligonucleotide primers withhomologous sequences in template double-stranded nucleic acid. In thisway, DNA synthesis is directed to defined points in a templatedouble-stranded nucleic acid. Using two or more sequence-specific (e.g.,gene-specific) primers, an exponential amplification reaction isinitiated if the template nucleic acid is present. The reactionprogresses rapidly and results in specific amplification of a sequencepresent within the template double-stranded nucleic acid from just a fewcopies of the template nucleic acid to detectable levels of theamplified products within minutes. RPA methods are disclosed, e.g., inU.S. Pat. Nos. 7,270,981; 7,399,590; 7,666,598; 7,435,561; US2009/0029421; and WO 2010/141940, all of which are incorporated hereinby reference.

The terms “templates” and “primers” are used interchangeably and refergenerally to an oligonucleotide sequence that serves as a starting pointfor nucleotide amplification of a target sequence using a polymerase.Templates are defined as oligonucleotides that bind to a recognitionregion of a target sequence and also contain a nicking enzyme bindingregion upstream of the recognition region and a stabilizing regionupstream to the nicking enzyme binding region. The compositionsdisclosed herein can contain a set of templates that amplify the targetnucleic acid sequence. The templates can comprise sequences that arecomplementary to the target nucleic acid sequence or that differ fromthe target nucleic acid sequence at one or more positions. Design oftemplates suitable for NEAR amplification methods disclosed herein areprovided in, for example, U.S. 2009/0081670 and U.S. 2009/0017453, eachof which are incorporated herein by reference.

The templates of the present methods may include, for example, spacers,blocking groups, and modified nucleotides. Modified nucleotides arenucleotides or nucleotide triphosphates that differ in compositionand/or structure from natural nucleotides and nucleotide triphosphates.Modifications include those naturally occurring that result frommodification by enzymes that modify nucleotides, such asmethyltransferases. Modified nucleotides also include synthetic ornon-naturally occurring nucleotides. For example, modified nucleotidesinclude those with 2′ modifications, such as 2′-O-methyl and 2′-fluoro.Other 2′-modified nucleotides are known in the art and are described in,for example U.S. Pat. No. 9,096,897, which is incorporated herein byreference in its entirety. Modified nucleotides or nucleotidetriphosphates used herein may, for example, be modified in such a waythat, when the modifications are present on one strand of adouble-stranded nucleic acid where there is a restriction endonucleaserecognition site, the modified nucleotide or nucleotide triphosphatesprotect the modified strand against cleavage by restriction enzymes.Thus, the presence of the modified nucleotides or nucleotidetriphosphates encourages the nicking rather than the cleavage of thedouble-stranded nucleic acid. Blocking groups are chemical moieties thatcan be added to the template to inhibit target sequence-independentnucleic acid polymerization by the polymerase. Blocking groups areusually located at the 3′ end of the template. Examples of blockinggroups include, for example, alkyl groups, non-nucleotide linkers,phosphorothioate, alkane-diol residues, peptide nucleic acid, andnucleotide derivatives lacking a 3′-OH, including, for example,cordycepin. Examples of spacers, include, for example, C3 spacers.Spacers may be used, for example, within the template, and also, forexample, at the 5′ end to attach other groups, such as, for example,labels.

In another aspect of the invention, there is provided a method fordetection of S. pyogenes in a sample comprising the steps of obtaining atissue sample from a subject; extracting nucleic acids from the sample;and amplifying the nucleic acid; wherein the nucleic acid is amplifiedand detected with templates and probes as described herein.

The present invention further comprises detecting the amplificationproduct, for example, by a method selected from the group consisting ofgel electrophoresis, mass spectrometry, SYBR I fluorescence, SYBR IIfluorescence, SYBR Gold, Pico Green, TOTO-3, intercalating dyedetection, FRET, molecular beacon detection, surface capture, capillaryelectrophoresis, incorporation of labeled nucleotides to allow detectionby capture, fluorescence polarization, and lateral flow capture.Amplification products can be detected, for example, by chemicalmoieties that intercalate into double-stranded DNA. For example, SYBRGreen® binds double-stranded DNA and upon excitation emit light; thus asproduct accumulates, fluorescence increases. The amplification productsmay be, for example, detected using a solid surface method, for example,where at least one capture probe is immobilized on the solid surfacethat binds to the amplified sequence. In some embodiments of allaspects, detecting the amplified product is performed using “real-timefluorescence.”

The term “real-time fluorescence,” refers to the detection of nucleicacid amplification products via a fluorescent signal generated by thecoupling of a fluorogenic dye molecule and a quencher moiety to the sameor different oligonucleotide substrates. Examples of commonly usedprobes are TAQMAN® probes, Molecular Beacon probes, and SCORPION®probes. Briefly, TAQMAN® probes, Molecular Beacons, and SCORPION® probeseach have a fluorescent reporter dye (also called a “fluor”) and aquencher moiety attached in close proximity to one another. In theunhybridized state, the proximity of the fluor and the quenchermolecules prevents the detection of fluorescent signal from the probe;in the case of TAQMAN® probes, during amplification, when the polymerasereplicates a template on which a probe is bound, the 5′-nucleaseactivity of the polymerase cleaves the probe thus, increasingfluorescence with each replication cycle as the fluor and quencher areseparated from one another. Molecular beacons probes emit fluorescencefollowing annealing of a homologous product, as this event induces aconformational change in the structure of the beacon, thereby separatingthe fluor and quencher. Briefly, TAQMAN® probesand SCORPION® probes,similar to Molecular beacons, will release fluorescence signal when aspecific product anneals to the probe and is extended, which leads toseparation of the fluor and quencher.

The production or presence of target nucleic acids and nucleic acidsequences may also be detected and monitored by lateral flow devices.Lateral Flow devices are well known. These devices generally include asolid phase fluid permeable flow path through which fluid flows throughby capillary force. Examples include, but are not limited to, dipstickassays and thin layer chromatographic plates with various appropriatecoatings. Immobilized on the flow path are various binding reagents forthe sample, binding partners or conjugates involving binding partnersfor the sample and signal producing systems. Detection of samples can beachieved in several manners; enzymatic detection, nanoparticledetection, colorimetric detection, and fluorescence detection, forexample. Enzymatic detection may involve enzyme-labeled probes that arehybridized to complementary or substantially complementary nucleic acidtargets on the surface of the lateral flow device. The resulting complexcan be treated with appropriate markers to develop a readable signal.Nanoparticle detection involves bead technology that may use colloidalgold, latex and paramagnetic nanoparticles. In one example, beads may beconjugated to an anti-biotin antibody. Target sequences may be directlybiotinylated, or target sequences may be hybridized to a sequencespecific biotinylated probes. Gold and latex give rise to colorimetricsignals visible to the naked eye and paramagnetic particles give rise toa non-visual signal when excited in a magnetic field and can beinterpreted by a specialized reader.

Nucleic acids can also be captured on lateral flow devices. Means ofcapture may include antibody dependent and antibody independent methods.Antibody-dependent capture generally comprises an antibody capture lineand a labeled probe that is complementary or substantially complementarysequence to the target. Antibody-independent capture generally usesnon-covalent interactions between two binding partners, for example, thehigh affinity and irreversible linkage between a biotinylated probe anda Strep Avidin line. Capture probes may be immobilized directly onlateral flow membranes. Both antibody dependent and antibody independentmethods may be used in multiplexing.

The production or presence of target nucleic acids and nucleic acidsequences may also be detected and monitored by multiplex DNAsequencing. Multiplex DNA sequencing is a means of identifying targetDNA sequences from a pool of DNA. The technique allows for thesimultaneous processing of many sequencing templates. Pooled multipletemplates can be resolved into individual sequences at the completion ofprocess.

The terms “complementary” and “substantially complementary” refer tobase pairing between nucleotides or nucleic acids, such as, forinstance, between the two strands of a double-stranded DNA molecule orbetween an oligonucleotide primer and a primer binding site on asingle-stranded nucleic acid to be sequenced or amplified. Complementarynucleotides are, generally, A and T (or A and U), and G and C. Withinthe context of the present invention, it is to be understood that thespecific sequence lengths listed are illustrative and not limiting andthat sequences covering the same map positions, but having slightlyfewer or greater numbers of bases are deemed to be equivalents of thesequences and fall within the scope of the invention, provided they willhybridize to the same positions on the target as the listed sequences.Because it is understood that nucleic acids do not require completecomplementarity in order to hybridize, the probe and primer sequencesdisclosed herein may be modified to some extent without loss of utilityas specific primers and probes. Generally, sequences having homology of80%, 90%, 95%, 97%, 98%, 99% fall within the scope of the presentinvention. As is known in the art, hybridization of complementary andpartially complementary nucleic acid sequences may be obtained byadjustment of the hybridization conditions to increase or decreasestringency, i.e., by adjustment of hybridization temperature or saltcontent of the buffer.

In some embodiments of all aspects, the template and probes can belabeled with (e.g., conjugated to) a detectable label. The term“detectable label” and “label” as used herein refers to any chemicalmoiety that can be used to provide a detectable (preferablyquantifiable) signal, and that can be attached to a nucleic acid orprotein via a covalent bond or noncovalent interaction (e.g., throughionic or hydrogen bonding, or via immobilization, adsorption, or thelike). Labels generally provide signals detectable by fluorescence,chemiluminescence, radioactivity, colorimetry, mass spectrometry, X-raydiffraction or absorption, magnetism, enzymatic activity, or the like.Examples of labels include haptens, enzymes, enzyme substrates,coenzymes, enzyme inhibitors, fluorophores, quenchers, chromophores,magnetic particles or beads, redox sensitive moieties (e.g.,electrochemically active moieties), luminescent markers, radioisotopes(including radionucleotides), and members of binding pairs. Morespecific examples include fluorescein, phycobiliprotein, tetraethylrhodamine, and beta-galactosidase. Binding pairs may includebiotin/Strepavidin, biotin/avidin, biotin/neutravidin,biotin/captavidin, epitope/antibody, protein A/immunoglobulin, proteinG/immunoglobulin, protein L/immunoglobulin, GST/glutathione,His-tag/Metal ion (e.g., nickel, cobalt or copper), antigen/antibody,FLAG/M1 antibody, maltose binding protein/maltose, calmodulin bindingprotein/calmodulin, enzyme-enzyme substrate, receptor-ligand bindingpairs, and analogs and mutants of the binding pairs.

As used herein, the terms “fluorescence label” and “fluorophore” areused interchangeably and refer to any substance that emitselectromagnetic energy at a certain wavelength (emission wavelength)when the substance is illuminated by radiation of a different wavelength(excitation wavelength) and is intended to encompass a chemical orbiochemical molecule or fragments thereof that is capable of interactingor reacting specifically with an analyte of interest in a sample toprovide one or more optical signals.

Representative fluorophores for use in the methods provided hereininclude, for example, FAM, (tetramethylrhodamine) Texas Red™, greenfluorescent protein, blue fluorescent protein, red fluorescent protein,fluorescein, fluorescein 5-isothiocyanate (FITC), cyanine dyes (Cy3,Cy3.5, Cy5, Cy5.5, Cy7), Bodipy dyes (Invitrogen) and/or Alexa Fluordyes (Invitrogen), dansyl, Dansyl Chloride (DNS-C1),5-(iodoacetamida)fluorescein (5-IAF,6-acryloyl-2-dimethylaminonaphthalene (acrylodan),7-nitrobenzo-2-oxa-1,3,-diazol-4-yl chloride (NBD-Cl), ethidium bromide,Lucifer Yellow, rhodamine dyes (5-carboxyrhodamine 6G hydrochloride,Lissamine rhodamine B sulfonyl chloride, rhodamine-B-isothiocyanate(RITC (rhodamine-B-isothiocyanate), rhodamine 800); tetramethylrhodamine5 -(and 6-)isothiocyanate (TRITC)), Texas Red™, sulfonyl chloride,naphthalamine sulfonic acids including but not limited to1-anilinonaphthalene-8 -sulfonic acid (ANS) and6-(p-toluidinyl)naphthalen-e-2-sulfonic acid (TNS), Anthroyl fatty acid,DPH, Parinaric acid, TMA-DPH, Fluorenyl fatty acid,Fluorescein-phosphatidylethanolamine, Texasred-phosphatidylethanolamine, Pyrenyl-phophatidylcholine,Fluorenyl-phosphotidylcholine, Merocyanine 540, Naphtyl Styryl,3,3′dipropylthiadicarbocyanine (diS-C3-(5)), 4-(p-dipentylaminostyryl)-l-methylpyridinium (di-5-ASP), Cy-3 lodo Acetamide,Cy-5-N-Hydroxysuccinimide, Cy-7-Isothiocyanate, IR-125, Thiazole Orange,Azure B, Nile Blue, Al Phthalocyanine, Oxaxine 1, 4′,6-diamidino-2-phenylindole. (DAPI), Hoechst 33342, TOTO, AcridineOrange, Ethidium Homodimer, N(ethoxycarbonylmethyl)-6-methoxyquinolinium(MQAE), Fura-2, Calcium Green, Carboxy SNARF-6, BAPTA, coumarin,phytofiuors, Coronene, and metal-ligand complexes.

It should be noted that a fluorescence quencher is also considered adetectable label. For example, the fluorescence quencher may becontacted to a fluorescent dye and the amount of quenching may bedetected.

Haptens for use in the methods provided herein include, for example,digoxigenin, glutathione and biotin.

Enzymes for use in the methods provided herein include, for example,alkaline phosphatase (AP), beta-galactosidase, horse radish peroxidase(HRP), soy bean peroxidase (SBP), urease, beta-lactamase and glucoseoxidase.

The compositions for detecting the presence or absence of S. pyogenes ina biological sample described herein, e.g., reagent mixtures, caninclude a DNA polymerase. The DNA polymerase disclosed herein may be aeukaryotic or prokaryotic polymerase. The polymerase may be selectedfrom, for example, Geobacillus bogazici DNA polymerase, Bst DNApolymerase, Bst DNA polymerase (Large fragment), 9° Nm DNA polymerase,Phi29 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I,Large, (Klenow) fragment, Klenow fragment (3′-5′ exo-), T4 DNApolymerase, T7 DNA polymerase, Deep VentR™ (exo-) DNA Polymerase, DeepVentR™ DNA Polymerase, DyNAzyme™ EXT DNA, DyNAzyme™ II Hot Start DNAPolymerase, Phusion™ High-Fidelity DNA Polymerase, Therminator™ DNAPolymerase, Therminator™ II DNA Polymerase, VentR® DNA Polymerase,VentR® (exo-) DNA Polymerase, RepliPHI™ Phi29 DNA Polymerase, rBst DNAPolymerase, rBst DNA Polymerase, Large Fragment (IsoTherm™ DNAPolymerase), MasterAmp™ AmpliTherm™ DNA Polymerase, Taq DNA polymerase,Tth DNA polymerase, Tfl DNA polymerase, Tgo DNA polymerase, SP6 DNApolymerase, Tbr DNA polymerase, DNA polymerase Beta, ThermoPhi DNApolymerase and Pyrophage 3173 (Lucigen), or combinations thereof.

The compositions for detecting the presence or absence of S. pyogenes ina biological sample described herein, such as reagent mixtures andbuffered solutions, can include an antimicrobial agent or preservative.An antimicrobial agent can inhibit the growth of microorganisms andincrease the shelf life of a reagent mixture or buffer solution. Theantimicrobial agent can include benzalkonium chloride,5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one,ProClin® (e.g., ProClin 300®, ProClin® 950, etc.), azides, merthiolates,and/or antibiotics. In some embodiments, the antimicrobial agentincludes 5-chloro-2-methyl-4-isothiazolin-3-one and2-methyl-4-isothiazolin-3-one.

By “constant temperature,” “isothermal conditions,” “essentiallyisothermal,” or “isothermally” is meant a set of reaction conditionswhere the temperature of the reaction is kept essentially orsubstantially constant during the course of the amplification reaction.An advantage of the amplification method of the present methods is thatthe temperature does not need to be cycled between an upper temperatureand a lower temperature. The nicking and the extension reaction willwork at the same temperature or within the same narrow temperaturerange. However, it is not necessary that the temperature be maintainedat precisely one temperature. If the equipment used to maintain anelevated temperature allows the temperature of the reaction mixture tovary by a few degrees, or few tenths of a degree, such as, for example,less than 1 degree, 0.8 degrees, 0.6 degrees, 0.4 degrees, or 0.2degrees, this is not detrimental to the amplification reaction, and maystill be considered to be an isothermal reaction.

The present invention may be used for multiplex amplification. Thus, forexample, in certain embodiments of the present invention at least twotarget sequences are capable of being amplified. The term “multiplexamplification” refers to the amplification of more than one nucleic acidof interest. By “capable of being amplified” is meant the amplificationreaction comprises the appropriate templates and enzymes to amplify atleast two target sequences. Thus, for example, the amplificationreaction may be prepared to detect at least two target sequences, butonly one of the target sequences may actually be present in the samplebeing tested, such that both sequences are capable of being amplified,even though only one sequence may actually be amplified. Or, where twotarget sequences are present, the amplification reaction may result inthe amplification of both of the target sequences. The multiplexamplification reaction may result in the amplification of one, some, orall, of the target sequences for which it comprises the appropriatetemplates and enzymes.

As used herein, the term “biological sample” or “sample” refers to asample obtained or derived from a subject (i.e., a patient). By way ofexample, the biological sample may be a tissue fluid obtained from asubject, which may be selected from the group consisting of blood,plasma, serum, lymphatic fluid, synovial fluid, cerebrospinal fluid,amniotic fluid, amniotic cord blood, tears, saliva, mucus secretions,urine and nasopharyngeal washes. Representative biological samples fromthe respiratory tract include wound and throat swabs, throat washings,nasal swabs, and specimens from the lower respiratory tract.

As used herein, “obtain” or “obtaining” can be any means whereby onecomes into possession of the sample by “direct” or “indirect” means.Directly obtaining a sample means performing a process (e.g., performinga physical method such as extraction) to obtain the sample. Indirectlyobtaining a sample refers to receiving the sample from another party orsource (e.g., a third party laboratory that directly acquired thesample). Directly obtaining a sample includes performing a process thatincludes a physical change in a physical substance, e.g., a startingmaterial, such as a blood, e.g., blood that was previously isolated froma patient. Thus, obtain is used to mean collection and/or removal of thesample from the subject. Furthermore, “obtain” is also used to meanwhere one receives the sample from another who was in possession of thesample previously.

The term “subject” refers to an animal or human, or to one or more cellsderived from an animal or human. Preferably, the subject is a human.Subjects can also include non-human primates. Cells may be in any form,including but not limited to cells retained in tissue, cell clusters,immortalized, transfected or transformed cells, and cells derived froman animal that has been physically or phenotypically altered. A humansubject can be known as a patient.

In yet other embodiments, a kit is provided for following the methods ofthe present invention for nucleic acid amplification, comprising a DNApolymerase; a first template for nucleic acid amplification, comprisinga recognition region at the 3′ end that is complementary orsubstantially complementary to the 3′ end of a target sequence antisensestrand; a nicking enzyme binding site and a nicking site upstream ofsaid recognition region; and a stabilizing region upstream of saidnicking enzyme binding site and said nicking site; a second template fornucleic acid amplification, comprising a recognition region at the 3′end that is complementary or substantially complementary to the 3′ endof a target sequence sense strand; nicking enzyme binding site and anicking site upstream of said recognition region; and a stabilizingregion upstream of said nicking enzyme binding site and said nickingsite; one or more nicking enzymes, wherein either one enzyme is capableof nicking at the nicking site of said first and said second templates,or a first enzyme is capable of nicking at the nicking site of saidfirst primer and a second enzyme is capable of nicking at the enzymesite of said second primer.

The kits used for the present methods may also comprise one or more ofthe components in any number of separate containers, packets, tubes,vials, microtiter plates and the like, or the components may be combinedin various combinations in such containers.

The components of the kit may, for example, be present in one or morecontainers, for example, all of the components may be in one container,or, for example, the enzymes may be in a separate container from thetemplates. The components may, for example, be lyophilized, freezedried, or in a stable buffer. In one example, the polymerase and nickingenzymes are in lyophilized form in a single container, and the templatesare either lyophilized, freeze dried, or in buffer, in a differentcontainer. Or, in another example, the polymerase, nicking enzymes, andthe templates are, in lyophilized form, in a single container. Or, thepolymerase and the nicking enzyme may be separated into differentcontainers.

Kits may further comprise, for example, dNTPs used in the reaction, ormodified nucleotides, cuvettes or other containers used for thereaction, or a vial of water or buffer for re-hydrating lyophilizedcomponents. The buffer used may, for example, be appropriate for bothpolymerase and nicking enzyme activity.

The kits used for the present methods may also comprise instructions forperforming one or more methods described herein and/or a description ofone or more compositions or reagents described herein. Instructionsand/or descriptions may be in printed form and may be included in a kitinsert. A kit also may include a written description of an Internetlocation that provides such instructions or descriptions.

Kits may further comprise reagents used for detection methods, such as,for example, reagents used for FRET, lateral flow devices, dipsticks,fluorescent dye, colloidal gold particles, latex particles, a molecularbeacon, or polystyrene beads.

An advantage of the present methods and the present kits is that theycan be used in any device that provides a constant temperature,including thermocyclers, incubation ovens, water baths, and heat blocks.

Methods using capture probes for detection include, for example, the useof a nucleic acid molecule (the capture probe) comprising a sequencethat is complementary to, or substantially complementary to, anamplification product strand such that the capture probe binds toamplified nucleic acid. The probe may be linked to a detectable label incertain embodiments, and amplification product may be detected based onthe detectable label of the probe specifically hybridized to theamplification product. The reaction may, for example, further comprisean antibody directed against a molecule incorporated into or attached tothe capture probe. Or, for example, the capture probe, or a moleculethat binds to the capture probe, may incorporate, for example, an enzymelabel, for example, peroxidase, alkaline phosphatase, orbeta-galactosidase, a fluorescent label, such as, for example,fluorescein or rhodamine, or, for example, other molecules havingchemiluminescent or bioluminescent activity. In some embodiments, theprobe is linked to a solid support, and amplification product strandsmay be specifically immobilized to the capture probe linked to the solidsupport under conditions known and selected by the person of ordinaryskill in the art. In the latter embodiments, a solid support-immobilizedamplification product may be subjected to processing steps, such aswashing, ion exchange, release from the solid support, or otherprocessing steps. An amplification product may be detected whenimmobilized to a solid support in some embodiments. The embodiments ofthe present invention also comprise combinations of these detection andanalysis methods.

In yet another embodiment, the methods further comprise modifying thesubject's clinical record to identify the subject as being diagnosed ashaving a S. pyogenes infection. Preferably, the clinical record isstored in a computer readable medium.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims. Those ofordinary skill in the art understand that for an exemplary assay,numerous modifications may be made to the volumes and format of thereaction, the length of time that the assay is conducted, and theamounts of each reactant.

NEAR of Streptococcus pyogenes Genomic DNA

Target Selection

A NEAR assay targeting GAS was developed that is unique to GAS whileshowing conservation across all strains and isolates of GAS found in thepublic domain at the time this analysis was performed. Initialbioinformatics analyses were performed using sequence data available atthe time from NCBI's Nucleotide Database(http://www.ncbi.nlm.nih.gov/nuccore) and Genome Database(http://www.ncbi.nlm.nih.gov/genome?db=genome). Multiple sequencealignments to identify regions of conservation were performed andmultiple regions within the GAS genome were strongly conserved. Allsequence segments that did show homology were subjected to BLASTanalysis to determine whether these sequences were unique to GAS.Following BLAST analyses, all sequences identified as unique to GAS wereused for template set generation (BLAST analysis performed using thenucleotide collection (nr/nt) database). Template sets were generatedusing an in-house automated software tool. In silico filtering was thenapplied to finalize the selection of template sets to screen in thelaboratory. In general, these filtering criteria involved analyzing thetemplate sets for potential interactions that could reduce assayperformance, including several parameters identified in-house that mightspecifically impact NEAR performance as well as parameters that arecommon to most nucleic acid amplification technologies. Using thesefiltering criteria, a final set of GAS template sets were selected forscreening.

The screening process resulted in the development of at least one GASassay.

The assay targets the cell envelope proteinase A (cepA) gene. This geneencodes for the interleukin-8 cleaving enzyme, which is used by S.pyogenes to inactivate the neutrophil chemoattractant interleukin-8. ThecepA 3062 assay targets position 345,952-345,994 of the GAS referencestrain M1 (NC 002737.1) and position 1,833-1,874 of the cepA gene. Theassay templates, probe (molecular beacon) and product sequences areprovided in Table 1. The probe is labeled with FAM reporter and BHQ-1quencher.

TABLE 1 Sequence ID (5′/3′) Oligo [ ] Template 1a- AGACTCCATATGG 500 nMSpy cepA s3062 F30 AGTCTAGCCAAAC AGGAACA (SEQ ID NO: 1) Template 2a-CGACTCCATATGGA 100 nM Spy cepA s3062 R41m GTCGaaAGCAATCT GAGGA(SEQ ID NO: 2) Template 1b- AGACTCCACACGGA 500 nM Spy cepA F30b.5omGTCTAGCCAAACAG mGmAmAmCmA (SEQ ID NO: 8) Template 2b- GGACTCCACACGGA100 nM Spy cepA R41m. GTCCGCCAGCAATC 1b.5om mUmGmAmGmG (SEQ ID NO: 9)Molecular beacon- ACAAGTATGTGAGG 200 nM Spy.cepA.S4.P1.MB4AGAGGCCATACTTG (Fam/BHQ1) T (SEQ ID NO: 3) Product 1 CAAACAGGAACAAGTATGGCCTCTCCTC AGATTGC (SEQ ID NO: 4) Product 2 GCAATCTGAGGAGAGGCCATACTTGTTC CTGTTTG (SEQ ID NO: 5)

To confirm that the targeted sequence was conserved among all GAS cepAsequences found in the public domain as well as unique to GAS, multiplesequence alignments and BLAST analyses were performed. Multiplealignment analysis of these sequences showed complete homology for theregion of the gene targeted by the 3062 assay. Further, there arecurrently 24 complete GAS genomes (including whole genome shotgunsequence) available for sequence analysis in NCBI Genome. The cepA geneis present in all 24 genomes, and the 3062 target region within cepA isconserved among all 24 genomes. Upon BLAST analysis, it was confirmedthat no other species contain significant homology to the 3062 targetsequence.

Assay Development

As a reference, the reagent mixtures discussed below are annotated asfollows: 25C—4× lyophilization mix, single tube assay format; 50C—2×lyophilization mix, single tube assay format; 25T—4× lyophilization mix,target assay only; 50T—2× lyophilization mix, target assay only; 25I—4×lyophilization mix, IC assay only; 50I—2× lyophilization mix, internalcontrol (IC) assay only.

The GAS NEAR assay can be run on an appropriate platform. For example,the GAS NEAR assay can be run on an Alere i platform(http://www.alere.com/ww/en/product-details/alere-i-strep-a.html). AnAlere i system consists of an instrument which provides heating, mixingand fluorescence detection with automated result output, and a set ofdisposables, consisting of the sample receiver (where the elution bufferis stored), a test base (containing two tubes of lyophilized NEARreagents) and a transfer device (designed to transfer 100 μl aliquots ofeluted sample from the sample receiver to each of the two tubescontaining lyophilized NEAR reagents located in the test base). Suitabledisposables for use with the Alere i GAS NEAR test include thosedescribed in, for example U.S. application Ser. No. 13/242,999,incorporated herein by reference in its entirety.

In addition to containing the reagents necessary for driving the GASNEAR assay, the lyophilized material also contains the lytic agent forGAS, the protein plyC; therefore, GAS lysis does not occur until thelyophilized material is re-suspended. In some cases, the lyophilizedmaterial does not contain a lytic agent for GAS, for example, in someexamples, the lyophilized material does not contain the protein plyC.The elution buffer was designed to allow for the rapid release of GASorganisms from clinical sample throat swabs as well as to provide thenecessary salts for driving the NEAR assay (both MgSO4 and (NH4)2SO4),in a slightly basic environment. In some examples, the elution bufferalso includes an anti-microbial agent or preservative (e.g., ProClin®950).

For the present examples, GAS assay was performed as a two tube assay—aGAS target specific assay in one tube, and an internal control (IC)assay in a second tube (tested side by side on the Alere i).

Assay Performance:

FIG. 1 shows the results of a limits of detection (LOD) study performedusing the GAS assay under various conditions. The LOD study wasperformed using a standard NEAR ‘hot start’ approach where the sampleand lyophilized mix were both pre-heated at 56° C. for 3 minutes andthen combined. Comparing the four mixes to one another, it is clear thatthe target only assays (25T & 50T) perform more robustly than thecombined or complete (25C & 50C) assays, especially at low copy number.From these data it was concluded that the LOD is 10-25 copies ofpurified genomic DNA for mixes 25T & 50T, 25 copies for mix 25C and 50copies for mix 50C, when a standard ‘hot start’ approach is used.

Internal Control (IC):

An IC assay was developed using a DNA oligonucleotide designed to serveas the IC target, and a molecular beacon was designed to specificallydetect the product generated off of the IC target (representativeoligonucleotides can be found in Table 2). This DNA oligonucleotidecontains 5′ and 3′ ends that are complementary to the target templateset's recognition regions but with a spacer region that differs from thetarget's spacer region. Thus, the same template set is used to amplifyboth target and IC.

TABLE 2 Internal Control (IC) oligonucleotideand molecular beacon sequences. Sequence Oligo ID (5′/3′) [ ]Internal control TGTAGCTG 100 nM molecular beacon- ACACCACC Flu BAAGCTACA ICMB4 Rox (SEQ ID (Rox/BHQ2) NO: 6) Internal control ACAATCTGA10,000 oligo-Spy GGAGCTGAC copies CepA.S4′.IC.3_11.11 ACCACCAAG 200,000Internal control CTACTGTTC copies oligo-Spy CTGTTTA CepA.S4′.IC_11.12(SEQ ID NO: 7) GCAATCTGAG GAGCTGACAC CACCAAGCTA CTGTTCCTGT TTGA (SEQ IDNO: 10)

FIG. 2 shows the performance of the IC assay when combined with thetarget assay (25C and 50C mixes), under standard NEAR ‘hot start’conditions. The results indicate robust IC performance in the presenceof GAS gDNA (from 0 to 250 copies of purified GAS gDNA). In allinstances, the 25C mix either outperformed or was equivalent to the 50Cmix.

Shown in FIG. 3 are the results of a similar experiment where the 25Iand 50I mixes were compared in the presence of 0-100,000 copies ofpurified GAS gDNA. The GAS IC assay showed robust performance in thepresence of a large amount of GAS gDNA, with no significant impact onperformance until 100,000 copies of GAS gDNA was present in thereaction. When no GAS target was present, both mixes showed strong ICamplification and detection. Further, up to 1000 copies of GAS targethad minimal if any impact on IC performance. Even at 10,000 copies ofGAS target the IC signal was still robust for both mix types. It was notuntil 100,000 copies of GAS target was added that a significantreduction in performance was found, and even at this copy numberamplification was still clearly well above background levels for boththe 25I and 50I mixes. Overall, the 25I showed slightly strongerfluorescence than the 50I mix.

Lyophilization:

In order to provide a viable point of care (POC) technology, thereagents used for GAS detection need to be stable for an extended periodof time at ambient temperature, or at minimum when stored at 2-8° C. Forexample, a minimum of six months storage at 2-8° C. without a loss inperformance is recommended, with the goal being 2-3 years. In order toprovide this level of stability, the NEAR assay reagents werelyophilized and subsequently packaged to minimize reagent exposure toboth moisture and oxygen. To accomplish this, the NEAR assay reagentsand lytic agent, plyC, were combined in a mix containing the excipientsdextrose and trehalose and subjected to freeze drying. Optimization ofboth the mix composition and freeze drying method were performed toyield a suitable lyophilized mix that retained activity while providinglong term stability. The final lyophilization mix and method selectedare shown below in Table 3. The GAS assay format provides for two tubes,one tube providing the target assay and a second tube providing the ICassay. These reagents are freeze dried using the same lyophilizationmethod and differ in composition only by the presence of the targetmolecular beacon in the target assay (& not in the IC assay) and the ICtarget oligo and IC molecular beacon in the IC assay (& not in thetarget assay). Both mixes are lyophilized as 2× (50 μl), meaning thatthe mixes are dried down at a 2× concentration and subsequentlyre-suspended in a 100 μl volume to provide a final 1× concentration ofeach reagent.

TABLE 3 GAS Lyophilization Mix - Reagent Composition Component v1.0 v2.0Strep A (Target) Lyo Conditions T1 500 nM F30 500 nM F30b.5om T2 100 nMR41m 100 nM R41m.1b.5om Target MB 200 nM MB4_FAM 200 nM MB4_Fam Pol(MG79) 3.0. ug 5.0 ug NE 30 U 0.7 ug ply C 1 ug 1 ug Tris pH 8.0 50 mM50 mM Dextran Dextran 150 Dextran 500 5% in 2x lyo 5% in 2x lyoTrehalose 100 mM in 2x lyo 100 mM in 2x lyo dNTPS 0.3 mM 0.3 mM Na₂SO₄15 mM 22.5 mM Triton X-100 0.10% 0.10% DTT 2 mM 2 mM Strep A (IC) LyoConditions T1 500 nM F30 500 nM F30b.5om T2 100 nM R41m 100 nMR41m.1b.5om IC 10,000 copies IC3_11.1 200,000 copies IC_11.12 IC MB 100nM IC_MB4_ROX 100 nM IC_MB4_ROX Pol (MG79) 3.0 ug 5.0 ug NE 30 U 0.6 ugply C 1 ug — Tris pH 8.0 50 mM 50 mM Dextran Dextran 150 Dextran 500 5%in 2x lyo 5% in 2x lyo Trehalose 100 mM in 2x lyo 100 mM in 2x lyo dNTPs0.3 mM 0.3 mM Na₂SO₄ 15 mM 22.5 mM Triton X-100 0.10% 0.10% DTT 2 mM 2mM

Shown in Table 3 are the final 1× reagent concentrations for the targetand IC assays. The lyophilized materials are freeze dried at a 2×concentration (50 μl volume) and subsequently re-suspended in a 100 μlvolume of elution buffer. In some cases, the two mixes are essentiallyidentically except that the target only mix does not contain ICcomponents and the IC only mix does not contain the target molecularbeacon. In some cases, the mixes differ in that one mix includes plyCand another mix does not, for example, in some cases the IC mix does notcontain plyC and the target mix contains plyC. In some cases, the mixesdiffer in the amount of NE, for example, in some cases the target mixcontains 0.7 μg of NE and the IC mix contains 0.6 μpg of NE.

GAS Lysis

For optimal performance, the GAS organism is lysed to gain access to thetarget nucleic acid and allow for specific amplification and detection.The streptococcal bacteriophage Cl lysin plyC is able to rapidly lysegroup A and C streptococci. Initial testing showed that the plyC proteinprovides rapid GAS bacterial lysis and further, that the presence of theprotein does not interfere with NEAR assay performance (data not shown).Subsequently, it was shown that the protein can be lyophilized incombination with NEAR assay reagents and re-suspended to provide rapid,in silico GAS lysis allowing for sensitive and specific NEAR GAS targetamplification and detection. To confirm that plyC can lyse GAS organismsunder NEAR reaction conditions, and that it is necessary for NEARdetection of GAS, a ‘hot start’ experiment was performed where thedetection of inactivated GAS organisms was tested plus or minus plyC(FIG. 4). In this experiment, 100 copies of inactivated GAS organismswere pre-heated to 56° C. and then combined with a mix of NEAR reagentscontaining 1 μg of plyC, and amplification was monitored for ten minutesusing a fluorescently labeled molecular beacon. As shown in FIG. 4, ifplyC was not present GAS was not detected (no fluorescent signal abovebackground), but the presence of plyC enabled the rapid and robustdetection of GAS, suggesting that the protein was able to readily lysethe target bacteria allowing access to the organism's genomic DNA.

Elution Buffer:

The GAS NEAR assay has been designed and developed to detect GAScollected from clinical throat swab samples. Each throat swab is elutedinto 2.5 ml of an optimized buffer (Table 4) following a two-minuteelution buffer pre-heat step (to reach a temperature of approximately40° C. on the Alere i). The swab is dipped into the buffer briefly,swirled, and then removed. The buffer enables rapid elution off of theswab while also providing: 1) Tris pH 8.0 to provide buffering capacity,2) the necessary salts to drive NEAR once it is used to re-suspend theNEAR lyophilized mix, 3) EDTA or EGTA to chelate any potential metalions that inhibit NEAR and 4) the detergent Triton X-100 which isbelieved to help in the elution process as well as to disperse cell/celldebris clumping. The buffer can also include an anti-microbial agent,for example ProClin® 950. As will be described in later sections of thisdocument, the elution buffer has been shown to effectively elute GASfrom throat swabs and also to enable NEAR amplification and detection.Throat swab samples containing GAS have been eluted using this bufferand the eluate has been subsequently used to re-suspend NEAR lyophilizedmaterial, resulting in the amplification and detection of GAS originallypresent on the throat swabs.

TABLE 4 Elution Buffer Composition Reagent v1.0 v2.0 Tris-Cl, pH 8.0 10mM 10 mM Triton X-100 0.10% 0.10% (NH₄)₂SO₄ 15 mM 15 mM MgSO₄ 15 mM 15mM Chelating agent 1 mM EDTA 1 mM EGTA Anti-microbial agent — 0.10%ProClin 950

LOD and Reproducibility Using Inactivated GAS Organisms:

To determine the LOD of the GAS assay (50T, target only assay) andensure its reproducibility, an experiment was performed to determine atwhich copy number the assay could detect 95% of the samples tested whentwenty replicates were screened. The study was performed on the Alere iusing inactivated GAS organisms (iGAS) at 25° C. with a 0 minute elutionbuffer pre-heat step. The results are shown in FIG. 5 and indicate thatthe 50T mix can readily detect 10 copies of iGAS even when the sampleand lyophilized material are not heated prior to combining. The datashow that 20/20 replicates were detected, although the fluorescencecurves did differ in both speed and intensity.

Impact of human genomic DNA (gDNA) on GAS assay.

One potential inhibitor of the NEAR technology is human gDNA. When athroat swab sample is collected from a patient symptomatic for GASinfection, it is possible that human gDNA is also collected on the swab(from immune cells such as white blood cells or from local epithelialcells). In order to assess the impact that human gDNA has on the GASassay, a study was performed using three different levels of GAS gDNA(25, 250 and 1000 copies) in the presence of 0, 10, 50, 100, 250, 500 or1000 ng of human gDNA. As shown in FIG. 6, the presence of human gDNAdoes have an impact on GAS assay performance, and the impact is GAStarget concentration dependent. When there is a low copy number oftarget GAS present in the reaction, 10 ng of human gDNA or moresignificantly inhibits the assay. At 250 copies of GAS target, theimpact of 10 ng of human gDNA is less, and at 1,000 copies of GAStarget, the effect of 10 ng of human gDNA on the assay is significantlyless. In fact, when 1,000 copies of target is present in the assay, upto 100 ng of human gDNA can be tolerated, albeit with a sloweramplification speed and reduced fluorescence signal. Testing of the 50I(IC only) mix showed a more robust response to human gDNA. When the 50Imix was tested in the prescence of 0 copies of target GAS and up to1,000 ng of human gDNA, the assay still produced a clearly positivesignal at 500 ng of human gDNA (even at 1,000 ng of human gDNA thefluorescence signal was still above background).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1.-47. (canceled)
 48. A composition comprising: a forward template comprising a nucleic acid sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end of the Streptococcus pyogenes (S. pyogenes ) cell envelope proteinase A (cepA) gene antisense strand; and a reverse template comprising a nucleotide sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end of the S. pyogenes cepA gene sense strand, wherein the forward template recognition region is 8-30 nucleotides in length and the reverse template recognition region is 8-30 nucleotides in length.
 49. The composition of claim 48, wherein the forward template further comprises a nicking enzyme binding site and a nicking site upstream of the forward template recognition region; and comprises a stabilizing region upstream of the nicking site.
 50. The composition of claim 48, wherein the reverse template further comprises a nicking enzyme binding site and a nicking site upstream the reverse template recognition region; and comprises a stabilizing region upstream of the nicking site.
 51. The composition of claim 48, further comprising a probe oligonucleotide comprising a sequence complementary to the S. pyogenes cepA gene nucleotide sequence.
 52. The composition of claim 48, further comprising a DNA polymerase; a nicking enzyme; and dNTPs or a mixture of dNTPs and ddNTPs.
 53. The composition of claim 48, wherein the composition is lyophilized.
 54. The composition of claim 48, wherein the forward template and/or the reverse template comprises a modified nucleotide, a spacer, or a blocking group.
 55. The composition of claim 48, wherein the forward template and/or the reverse template comprises a modified nucleotide comprising a 2′ modification.
 56. The composition of claim 48, wherein the forward template and/or the reverse template comprises a modified nucleotide comprising a 2′-O-methyl.
 57. The composition of claim 48, wherein the forward template and/or the reverse template comprises a phosphorothioate. 